The CD94/NKG2 receptors expressed by subpopulations of NK cells and T cells have been implicated as receptors for a broad range of both classical and nonclassical HLA class I molecules. To examine the ligand specificity of CD94/NKG2 proteins, a soluble heterodimeric form of the receptor was produced and used in direct binding studies with cells expressing defined HLA class I/peptide complexes. We confirmed that CD94/NKG2A specifically interacts with HLA-E and demonstrated that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2A and classical HLA class I molecules were observed, as assayed by direct binding of the soluble receptor or by functionalassays using CD94/NKG2A+ NK cells. The role of the peptide associated with HLA-E in the interaction between HLA-E and CD94/NKG2A was also assessed. All class I leader sequence peptides tested bound to HLA-E and were recognized by CD94/NKG2A. However, amino acid variations in class I leader sequences affected the stability of HLA-E. Additionally, not all HLA-E/peptide complexes examined were recognized by CD94/NKG2A. Thus CD94/NKG2A recognition of HLA-E is controlled by peptide at two levels; first, peptide must stabilize HLA- E and promote cell surface expression and second, the HLA-E/peptide complex must form the ligand for CD94/NKG2A. The crystal structure of the intracellular domain of CD94 was determined revealing a unique C- type lectin fold. The crystal structure is likely a prototype for other NK cell receptors such as Ly-49, NKR-P1 and CD-69.In primary embryonal fibroblasts from transgenic mice expressing H-2(b) genes and a miniature swine class I transgene (PD1), transformation with adenovirus 12 results in suppression of assembly and cell surface expression of all class I complexes. Cell surface expression of PD1 can be recovered by transfecting the cells with peptide transporter genes. However, reconstitution of the H-2K(b) gene expression requires, in addition, a 2-fold increase in the steady state level of the H-2K(b) mRNA that can be attained by treatment of the cells with interferons or by transfecting them with the H-2K(b) gene. A detailed analyses of the biogenesis of class I molecules has revealed the steady state expression of free class I heavy chains that are not converted into conformed complexes even when peptide transporter genes are overexpressed. - Natural Killer (NK) cells, HLA class I receptors, HLA- E, CD94/NKG2, MHC class I complexes, peptide binding, cancer